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Nucleic Acids Research, 1985, Vol. 13, No. 3 745-761
© 1985

Articles

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin

Anthony C Forster, James L. Mclnnes, Derek C. Skingle and Robert H. Symons*

Adelaide University Centre for Gene Technology, Department of Biochemistry. University of Adelaide Adelaide, South Australia 5000, Australia

*To whom correspondence should be addressed

Received November 15, 1984. Revised January 17, 1985. Accepted January 17, 1985.

A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N' (N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3-propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100–400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 x 10–18 mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32P-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.


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